Organ-specific transcriptional regulation by HFR1 and HY5 in response to shade in Arabidopsis

Light is crucial for plants and serves as a signal for modulating their growth. Under shade, where red to far-red light ratio is low, plants exhibit shade avoidance responses (SAR). LONG HYPOCOTYL IN FAR-RED 1 (HFR1) and ELONGATED HYPOCOTYL 5 (HY5) are known to be negative regulators of SAR and physically interact with one another. However, transcriptional regulatory network underlying SAR by these two transcription factors has not been explored. Here, we performed organ-specific transcriptome analyses of Arabidopsis thaliana hfr1-5, hy5-215 and hfr1hy5 to identify genes that are co-regulated by HFR1 and HY5 in hypocotyls and cotyledons. Genes co-regulated by HFR1 and HY5 were enriched in various processes related to cell wall modification and chlorophyll biosynthesis in hypocotyls. Phytohormone (abscisic acid and jasmonic acid) and light responses were significantly regulated by HFR1 and HY5 in both organs, though it is more prominent under shade in cotyledons. HFR1 and HY5 also differentially regulate the expression of the cell wall-related genes for xyloglucan endotransglucosylase/hydrolase, expansin, arabinogalactan protein and class III peroxidase depending on the organs. Furthermore, HFR1 and HY5 cooperatively regulated hypocotyl responsiveness to shade through auxin metabolism. Together, our study illustrates the importance of the HFR1-HY5 module in regulating organ-specific shade responses in Arabidopsis.

Seedlings of hfr1hy5, and cry1 were grown in continuous far-red (FR), red (R) or blue (B) light under different intensities for 4 d.The hypocotyl lengths of the seedlings were measured and plotted against various intensities.
Supplementary Figure 3. Influence of HFR1 and HY5 on phytochrome expressions.qPCR of phyA and phyB expression in 7-d-old Col-0, hfr1-5, hy5-215 and hfr1hy5 seedlings under light (L) and 1 h shade (S) treatment.Student's t-test was performed to determine significant differences compared to Col-0, within the same treatment group: *, P < 0.05.-log 10 P-value number of genes response to light stimulus cellular catabolic process response to light intensity lipid biosynthetic process organonitrogen compound catabolic process cellular nitrogen compound biosynthetic process response to cold photosynthesis macromolecular complex assembly response to organic cyclic compound -log 10 P-value number of genes response to wounding defense response to bacterium response to water deprivation response to salicylic acid cellular response to hypoxia response to heat response to oxidative stress shoot system development response to jasmonic acid response to abscisic acid

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Characterization of hfr1hy5 double mutant under monochromatic light.

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Additional phenotypic characterization of hfr1hy5 double mutant.(A)Percentage of hypocotyl increment from light to shade in Col-0, hfr1-5, hy5-215, hfr1hy5 seedlings from Figure1B.Total (B) chlorophyll and (C) carotenoid content of hfr1hy5   seedlings grown under light (L) and shade (S).Student's t-test was performed to determine significant differences compared to Col-0, within the same treatment group; *, P < 0.05.Influence of HFR1 and HY5 on their individual expressions.HFR1 and HY5 expression in 7-d-old Col-0, hfr1-5, hy5-215 seedlings under light (L) and 1 h shade (S) treatment.Student's t-test was performed to determine significant differences compared to Col-0, within the same treatment group; *, P < 0.05.DEG analysis of shade-responsive genes regulated by HFR1 and HY5.(A) Distribution of DEGs in 1 h shade-treated Col-0, hfr1, hy5 and hfr1hy5 hypocotyl and cotyledon samples compared to the respective light-treated genotypes.DEGs obtained from each genotype were plotted separately as up-regulated (pink, adjusted P ≤ 0.05 and log 2 fold change ≥ 1), or down-regulated (blue, adjusted P ≤ 0.05 and log 2 fold change ≤ -1).(B) Dot plot of GO terms from up-(pink) and down-regulated (blue) DEGs from (A).Dot sizes correspond to number of genes while the dot color represents the -log 10 Pvalue.signaling pathway response to gibberellin response to cytokinin response to salicylic acid salicylic acid mediated signaling pathway response to jasmonic acid jasmonic acid mediated signaling pathway jasmonic acid biosynthetic process response to abscisic acid abscisic acid-activated signaling pathway shade avoidance response to red light response to far red light response to red or far red light response to light stimulus response to blue light response to light intensity photomorphogenesis response to UV-B chloroplast organization chlorophyll biosynthetic process anthocyanin-containing compound biosynthetic process pigment GO analysis of genotype-specific DEGs under light and shade.Dot plot of GO terms enriched in up-(pink) and down-regulated (blue) DEGs from genotype-specific DEGs under (A) light and (B) shade.Dot sizes correspond to number of genes while the dot color represents the -log 10 P-value.JA and ABA content in hypocotyls under shade.Quantification of JA and ABA content extracted from hypocotyls of 7-d-old Col-0, hfr1-5, hy5-215 and hfr1hy5 seedlings under light (L) and 1 h shade (S).Student's t-tests were performed to determine significant differences compared to Col-0, within the same treatment group; *, P < 0.05.Identification of DEGs targeted by HFR1 and HY5 in cotyledons under shade.(A) Venn diagrams display the overlap between HY5 target genes obtained from HY5 ChIPseq data (Burko et al., 2020) and the up-regulated (left) or down-regulated (right) DEGs found in the cotyledons.Overlapped DEGs that were (B) up-regulated or (C) down-regulated under shade were categorized into nine different clusters through K-means clustering.Expression changes in y-axis represent Z-score of log 2 -fold change of the DEGs.The thin grey lines represent the individual genes in each cluster.The thick lines (red or blue) represent the centroids of each clusters.

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Differentially expressed auxin biosynthesis genes under light and shade.Heatmap of auxin biosynthesis gene expressions under (A) light and (B) shade in Col-0, hfr1, hy5 and hfr1hy5 cotyledon and hypocotyl samples.Gene expressions are displayed as Z-scores.